Easypure quick gel extraction kit

In order to determine the full-length genome of the CanineCV strain from Argentina, a positive sample was tested with the additional primer sets listed in Table 1 and sequenced twice. Resulting amplicons were eluted after agarose gel electrophoresis with Easy Pure Quick gel Extraction kit (TransGen Biotech) and submitted twice for sequencing to Unidad Genómica, Instituto de Biotecnología (INTA, Argentina) and to GenBiotech (Buenos Aires, Argentina), following the dideoxy-nucleotide chain termination method using the primers described above. The full-length genome was assembled using the Mafft software (EMBL-EBI). The complete sequence was named strain UBA-Baires and deposited in Genbank under accession number MK033608.

An inverse PCR method was employed to identify the flanking sequence of the inserted mini-Tn5 transposon in the genomes of the mutants [28 (link)]. Briefly, the total genomic DNAs of C. werkmanii BF-6 mutants were extracted from 1 mL of LB medium cultures using the EasyPure Bacteria Genomic DNA Kit (Transgen Biotech, Beijing, China). The isolated genomes were digested with TaqI (NEB, Beijing, China), self-ligated with T4 DNA ligase (NEB) and then used as templates for inverse PCR using the primers TnI-F and TnI-R (Table 2). The amplified fragments were separated by agarose gel electrophoresis, purified using the EasyPure Quick Gel Extraction Kit (Transgen Biotech, Beijing, China) and finally sequenced at the Invitrogen (Guangzhou, China). To verify the mutant genes, the obtained sequences were used to search the nucleotide databases developed by the National Center for Biotechnology Information (NCBI) and the available C. werkmanii BF-6 genome using the BLAST tool.

According to the predicted mRNA sequence of the ovine GLOD4 gene in the National Center for Biotechnology Information (NCBI), primers were designed using Primer Premier 6.0 (Premier Company, Canada) (Table 1). The mixed cDNA samples for testicular tissues derived from all rams were used as a template to amplify the CDS sequence of the GLOD4 gene. The PCR reaction system (25 μL) was comprised of 1 μL of cDNA, 0.8 μL of forward primer, 0.8 μL of reverse primer, 12.5 μL of 2 × Easy Taq PCR SuperMix (TransGen Biotech, Beijing, China), and 10 μL of ddH2O. Reaction procedures were as follows: 1 cycle of 95 °C for 4 min; 40 cycles of 94 °C for 45 s, 64 °C for 30 s; and 1 cycle of 72 °C for 90 s. The PCR product was separated using 1% agarose gel and purified with an EasyPure Quick Gel Extraction kit (TransGen Biotech, Beijing, China). The purified PCR products were ligated to the pEASY-Blunt vector using the pEASY-Blunt Cloning kit (TransGen Biotech, Beijing, China), and then transformed into Trans1-T1 competent cells (TransGen Biotech, Beijing, China) according to operating instructions. The three independent positive clones were selected and sequenced by the Qingke Biological Company (Xi’an, China).

To construct the Dmd minigene, exons 50, 51, and 52 of the mouse Dmd gene were amplified by PCR. The PCR products were purified using the EasyPure Quick Gel Extraction Kit (Transgen #EG101-01) and constructed into the pcDNA 3.1 vector through HindIII (New England Biolabs (NEB) #R3104) and EcoRI (NEB #R3101) restriction site using the ClonExpress II One Step Cloning Kit (Vazyme #C112). To construct a vector expressing linear AS-RNA, the PGL3 vector (Addgene #107721) was linearized by BsaI (NEB #R3733) and EcoRI (NEB #R3101). Antisense sequences of less than 50 base pairs (bp) were chemically synthesized, annealed, and ligated to the linearized PGL3 vector by T4 DNA ligase (Vazyme #C301). Antisense sequences greater than 100 bp were amplified by PCR and ligated to the linearized PGL3 vector using the ClonExpress II One Step Cloning Kit (Vazyme #C112). The transcription of AS-RNA was driven by the U6 promoter. DNA, including a Twister P3 U2A, a 5′ ligation sequence, a 3′ ligation sequence, and a Twister P1, was synthesized and cloned into a linearized PGL3 vector to construct the AS-circRNA expression vector. Antisense sequences of different lengths were ligated between the 2 ligation sequences by the method described above. The sequence of U7 has been published previously [10 (link)]. Primers and antisense sequences are listed in Table S2.

DNA methylation by bisulfite sequencing Genomic DNA was extracted from cells using DNeasy & Blood Tissue Kit (Qiagen) according to the manufacturer’s instructions. Bisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (Qiagen). Bisulfite converted DNA was amplified by seminested PCR, using HS EX Taq DNA Polymerase (Takara). Primer sequences are detailed in S3 Table. PCR products were recovered from stained gels (EasyPure Quick Gel Extraction Kit,Transgen), cloned into a pEASY-T1 Simple Cloning vector (Transgen) and then sequenced.

For the isolation of potential viral pathogens infecting tobacco plants, the symptomatic leaf samples were collected from Luzhou, Sichuan province, China in 2018. Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian, China) from tobacco leaves following the manufacturer’s guidelines. Viruses were detected by RT-PCR with four pairs of degenerate primers [27 ], including CMVCPf/CMVCPr (for cucumber mosaic virus), Tob-Uni1/Tob-Uni2 (for tobamoviruses), PotyF/PotyR (for potyviruses) and TSWVf/TSWVr (for tomato spotted wilt virus) (Additional file 1: Table S1). The amplified PCR products were purified with EasyPure Quick Gel Extraction kit (Transgen Biotech, Beijing, China), cloned into pEASY-T1 Cloning Vector (Transgen Biotech) and directly sequenced (Sangon Biotech, Shanghai, China), respectively.

The amplified CtCYP51 full-length fragments (primers are listed in Supplementary Table S3) were purified using the EasyPure Quick Gel Extraction Kit (TransGen, Beijing, China) and were cloned into the pEASY-T1 simple plasmid (TransGen, Beijing, China) according to the manufacturer’s recommendations. The vector that inserted into Escherichia coli strain was sequenced (Sunbiotech Co., Beijing, China) using vector primers M13F (5′-ACTGGCCGTCGTTTTAC-3′) and M13R (5′-GTCCTTTGT CGATACTG-3′) (Pang et al., 2013 (link); Fan et al., 2014 (link)). DNA sequences were analyzed with DNAMAN5.2.2.0 (Lynnon Biosoft, Quebec, QC, Canada).